The present invention relates to the production of soybean plants via tissue culture techniques, and more specifically, to a method of regenerating soybean plants from cultured soybean cotyledonary nodes.
Soybean, Glycine max, is an important annual legume whose seeds are valuable for the production of oil and protein. Despite extensive effort, researchers have had limited success in regenerating soybean plants from tissue culture and the methods which have been developed for regeneration of soybeans from tissue culture have little practical utility. This is unfortunate since in vitro tissue and cell culture methods are potentially useful tools in soybean breeding programs, particularly for the multiplication of hybrid germplasm.
In the case of plant regeneration of soybean, only limited success has been reported from either callus or cell suspension culture. Cheng, et al., PLANT SCIENCE LETTERS, 19:91-99 (1980) reports a technique for soybean plant regeneration from cotyledonary node segments. The method involves germinating soybean seeds for approximately four weeks on a modified Gamborg media containing sucrose as the carbon source. The basal media was supplemented with 0.025 .mu.M indole-3-butyric acid (IBA) and benzylaminopurine (BAP) at a concentration of from 0.2 .mu.M to 20 .mu.M. The cotyledonary node segments were prepared from the cultured soybean seedlings by removing the cotyledons adjacent to the stem axis, and cutting the stem approximately 3 mm above and below the node region. The cotyledonary node segments excised from the conditioned seedlings were cultured for four weeks on Gamborg medium supplemented with 0.025 .mu.M IBA and from 0.2 .mu.M to 20 .mu.M BAP and then transferred to a medium with 1.0 .mu.M BAP. At low BAP concentrations (i.e., less than 5.0 .mu.M), the extent of stimulation of shoot-bud formation differed among the tested soybean cultivars. The authors found that conditioned cotyledonary node segments derived from seedlings cultured with a high BAP concentration were most advantageous for stimulation of shoot-bud formation.
Cheng, et al., concluded that a high concentration of BAP is effective in stimulating bud formation but that it inhibits bud growth. When Cheng, et al., subsequently rooted shoots excised from cotyledonary node cultures of cultivars of Dare and Amsoy soybean cultures, the shoots rooted on basal media without addition of auxin. However, while the plants form the cultivar Dare appeared normal, plants from the cultivar Amsoy were abnormal in that the roots were enlarged in their upper regions and were covered with hairlike white cells and split to the core. Further, Cheng et al., often had difficulty in getting the shoots to root at all.
Phillips and Collins, 1981, reported a system of induction and development of somatic embryos from cell suspension cultures of soybean. The suspension cultures were used as inoculum sources for growing callus on agar-solidified nutrient media. The authors reported that numerous embryoids, particularly of Glycine soja, were produced on basal media supplemented with 100 ppm casein hydrolysate, 0.1 .mu.M abscisic acid, 2.25 .mu.M 2,4-dichlorophenoxyacetic acid, and 15.0 .mu.M adenine or 0.46 .mu.M kinetin. Callus recovered from the suspension culture produced one shoot structure when grown on a solid medium containing 0.2 .mu.M Amo 1618, an inhibitor of gibberellin synthesis and 80.0 .mu.M glutathione. The shoot structure consisted of two distinct buds, one producing two leaves. The shoot did not develop into a plant, thus, regeneration of soybean plants was not achieved.
Unlike the prior art methods which yield either no regeneration of soybean plants or regeneration at very low frequencies, often with abnormal soybean plants produced, the method of soybean regeneration described herein offers an efficient, high frequency method which results in the production of normal, healthy soybean plants.
Through the use of the method described herein, one is able to clone multiple individual soybean plants from a single mother plant, e.g., a hybrid (F1). Because the progeny are apparently genetically identical to the mother plant, many transplantable individuals (about 70 to 170), rather than a single individual, may be realized for earlier field evaluation of soybean hybrids. The need for the sexual cycle is totally negated and, thus, no genetic segregation will occur. This, in turn, negates the need for "roguing" of abberant individuals in the field. The procedure thus affords a 30 to 100 fold time savings in manual crossing and deletes one generation of the seed increase process. In the F2 generation, approximately 35,000 individuals can be evaluated, compared to 100 (assuming a single field-grown plant produced a total of 100 seeds). Total time saved in the breeding and hybrid evaluation time cycle could be at least five years.
Another use of the method of this invention is long-term unique germplasm preservation, including all of the ramifications of continuing to produce individuals with identical genotypes over a relatively long period, thus allowing multiple evaluations. Through the use of the procedure described herein, soybean regeneration has been observed from tissue culture for more than ten months. At the present time, nodal tissue has not been maintained in culture past ten months; however, it is expected that the tissue could be maintained in culture for extended periods of time while retaining regeneration capacity.
It will be apparent to skilled artisans that the method of this invention may be used for a variety of purposes. For example, the method described herein may be advantageously employed in a screening program directed to identifying and evaluating chemicals for biological activity in soybeans. Because the method yields shoots, buds, plantlets, and finally plants, chemicals may be screened in vitro at various stages of soybean development. Since the screening is done in vitro rather than in vivo, variability associated with uncontrolled environmental factors may be reduced as well as cost and time necessary for field testing.
Certain of the terms used in this patent specification are defined below; the definitions are believed to be the ones most commonly used by workers skilled in this art.
A "bud" is an undeveloped shoot comprised of a meristem which gives rise to a shoot.
A "cotyledon" is a part of the embryo and is a fleshy specialized leaf-like storage organ which provides nutrients during early seed germination.
A "cotyledonary node" is that part of the seedling including the embryonic axis to which the cotyledons are attached and which botanically defines the division of the hypocotyl and the epicotyl.
"Callus" is a mass of growing cells which may contain organized cells or cells at various stages of cytodifferentiation.
A "cytokinin" is a substance which, in combination with auxin, stimulates cell division in plants and which interacts with auxin in determining the direction which differentiation of the cell takes. All known naturally occurring cytokinins are adenine derivatives (i.e., they are 6-substituted amino-purines).
An "explant" is a piece of tissue taken from a donor plant which in culture will often produce a callus.
An "epicotyl" is the portion of the seedling above the cotyledons which develops into the shoot and its derivatives.
A "hypocotyl" is that portion of the stem of a plant embryo or seedling below the cotyledons and above the root.
A "meristem" is the undifferentiated, mitotically active tissues of a plant.
A "shoot" is derived from meristematic tissue (apical, lateral, or adventitious) and produces the above-ground portion of the plant.